豚鼠过敏抗体的分离纯化及在PCA试验中作用规律的初步研究

目的 建立豚鼠Ⅰ型过敏反应模型,对致敏血清中过敏抗体(IgE、IgG)进行初步分离纯化,并探讨过敏抗体的作用特点.方法 以卵白蛋白(OVA)和Al(OH)_3作为免疫原制备豚鼠Ⅰ型过敏反应模型,ELISA法检测抗体含量,用饱和硫酸铵沉淀、Protein A柱亲和层析的方法 对致敏血清中IgE、IgG抗体进行初步分离纯化,以豚鼠连续被动皮肤过敏试验分别观察两种抗体在致敏后不同时间激发时引起皮肤蓝斑的变化情况.结果 模型组和对照组血清中IgE抗体含量分别为719.3750ng/ml、2.5250 ng/ml,IgG抗体吸光度(A)值分别为0.9921、0.0174,模型组两种抗体含量均比对照组显著升高(P<0.05).豚鼠9 d连续被动皮肤过敏试验中,IgE抗体在5 d PCA时产生的蓝斑直径达到最大,之后持续存在;IgG抗体在2 d PCA时产生蓝斑直径最大,之后迅速下降.结论 成功将致敏血清中两种过敏抗体进行了初步分离纯化,IgE和IgG抗体均参与了豚鼠Ⅰ型过敏反应,IgG抗体较IgE抗体可能作用更为迅速、短暂,有可能成为疫苗Ⅰ型过敏反应毒理学评价的另一主要指标.

Isolation and purification of anaphylactic antibody from guinea pig and preliminary research on its role in passive cutaneous anaphylaxis test (PCA)

Objective To establish guinea pig model of type Ⅰ anaphylaxis, isolate the anaphy-lactic antibody(IgE and IgG) preliminarily from sera of sensitized guinea pigs, and investigate its functional characteristics. Methods Animal model was established by sensitizing guinea pigs with OVA and Al(OH)_3, level of antibody was determined by ELISA, IgE and IgG in sera were preliminarily isolated through saturated ammonium sulphate precipitate and affinity chromatograph of Protein A. A continuous passive cutaneous ana-phylaxis test (PCA) was performed by sensitizing guinea pigs individually with IgE and IgG and then challenging at different time , and the variation of blue spots in skin were observed after challenge . Results Concentration of IgE in model group and control group were 719.3750 ng/ml and 2.5250 ng/ml, the optical density of IgG in model group and control group were 0.9921 and 0.0174, the level of two antibodies in model group were significantly higher than that of control group (P<0.05). In 9 d continuous PCA test, the blue spot induced by IgE in skin lasted for 9 days and appeared the largest when challenged at day 5. The diameter of blue spot induced by IgG was the largest when challenged at day 2 and then decreased fast. Con-clusion Anaphylactic antibodies were successfully preliminarily isolated from sera of sensitizing guinea pig, both IgE and IgG play roles in type Ⅰ anaphylaxis of guinea pig, the hypersensitive reaction induced by IgG is fast and short than that induced by IgE, and IgG may become an important surrogate marker in immunotoxic-ity evaluation(type Ⅰ anaphylaxis)of vaccine.